From 9/7/2018 through 9/2/2019 Feldman supported zebrafish research of eleven labs and also conducted independent research. Porter Lab (NICHD): Genetic Dissection and Creation of Human Disease Models of Sterol Metabolism. In previous years the Core used CRISPR-Cas9 technology to create genetic mutant zebrafish lines for the Porter lab in four genes npc1, npc2, cln3 and ebp - with roles in various steps of cholesterol metabolism. In the previous year some phenotypes of npc1 mutants were characterized and published and this year other npc1 phenotypes have been under investigation as well as phenotypes of cln3 and ebp mutants. The Core also re-generated mutant alleles of dhcr7 and cln3 in the TAB5 genetic background with the goal of reducing phenotypic variability seen in the more outbred EK genetic background that was originally used. Stratakis Lab (NICHD): Function of Zebrafish Orthologs to Human Genes Implicated in Disorders of the Pituitary-Adrenal Axis. In previous years, the Core used CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function mutations in four zebrafish orthologs to human genes implicated by the Stratakis lab in human growth anomalies and eight zebrafish orthologs to human adrenal hyperplasia and Cushing disease-associated genes. Characterization of one of these genes has made considerable progress over the last year, with new findings of temperature-sensitive maternal effects on viability and CNS anatomy. Blackshear lab (NIEHS): Assessing Functions of a Zinc-Finger Protein Gene Family in Zebrafish. The Core has been engaged over the last several years to use CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function in seven zebrafish orthologues and assist with preliminary phenotype characterization. Phenotypes for fourteen mutant alleles for all seven genes have proven to be weak or absent. This year the core sent tissue samples to the Blackshear lab for comparing the transcriptomes of mutants versus control siblings in case subtle molecular consequences of these gene disruptions can illuminate their function. Eleven of the thirteen alleles have been cryopreserved and cryopreservation of the remaining three alleles is planned Larson Lab (NCI). This year the Core was engaged by the Larson lab to generate transgenic zebrafish carrying mutant and WT Runx1-mKate protein fusions. Optimal microinjection conditions for two constructs (third construct pending) have been determined, Swartz Lab (NINDS). This year the Swartz lab joined the core with the goal of seeing whether zebrafish tmem266 is expressed in the cerebellum. Whole-mount in situ hybridization experiments are ongoing. Olivier Lab (NHBLI). The Core began providing embryos to the Olivier lab this year, enabling them to conduct experiments on infection in their own laboratory and on their own animal protocol. Meilleur lab (NINR): Investigating the function of ryr1b mutants. Dr. Meilleurs team spent the first part of the year continuing previous efforts to optimize an assay to test candidate drugs for their ability to potentially ameliorate muscle defects seen in zebrafish mutants that carry mutations in ryr1b, a gene whose human counterpart is implicated in various myopathies. Due to technical challenges and a desire for a different genetic model, the drug testing component of this project was placed on hold, and the focus has shifted to creating new genetic models of ryr1b alleles. Caldovic Lab (Childrens National Medical Center): Finding Neuroprotective Drugs to Mitigate Hyperammonemia, a Consequence of Urea Cycle Defects & Liver Failure. Exposure of the brain to high ammonia causes neurcognitive deficits, intellectual disabilities, coma and death. Since 2012, the Core has helped this lab to use zebrafish embryos to identify small molecules able to diminish the effects of hyperammonemia. In the initial few years, a library of hundreds of small molecules with known safety profiles for humans was screened and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. A manuscript summarizing this work is currently being drafted. Over the last three years, the Core has supported a re-implementation of this screen using a larger library of 10,000 compounds, bolstered by additional personnel from the Tuchman lab. The screen was completed during the last year and the team has been focused on writing up the data and conducting validation assays. Independent Research by the NICHD Zebrafish Core: optimizing strategies for CRISPR/Cas9-based homology-directed repair (HDR). In the previous year, the Core used CRISPR-Cas9 technology in combination with donor DNA to generate a zebrafish line with an atp7a amino acid (AA) substitution that is cognate to an ATP7A (AA) substitution of interest to the Kaler lab (NICHD) that causes distal motoneuropathy. The phenotype is characterized by hypopigmentation, with no obvious diminishment of motor function. We devised a novel screening strategy for this project that included synonymous changes alongside the targeted non-synonymous change. These synonymous changes introduced RFLPs amenable to our molecular screening strategy. Over the past year, in collaboration with the NICHD Bioinformatics & Scientific Programming Core, software for generalizing this approach to any locus of interest has been developed. The Core has also been conducting Directors Award-funded research this year in collaboration with the NICHD Molecular Genomics Core to compare precise genome editing methods and efficiencies. Our initial comparison examines the efficiency of five distinct methods to generate seven distinct alleles of interest to labs in NICHD and elsewhere at NIH, namely: two npc1 alleles (Porter lab - NICHD), one rhoaa allele (Weinstein lab - NICHD), one ifitm5 allele (Marini lab - NICHD), two ryr1b alleles (Meilleur lab - NINR) and one cacna1c allele (Golden lab NIDDK).